Affirming NIH's commitment to addressing structural racism in the biomedical research enterprise.

NIH has acknowledged and committed to ending structural racism. The framework for NIH's approach, summarized here, includes understanding barriers; developing robust health disparities/equity research; improving its internal culture; being transparent and accountable; and changing the extramural ecosystem so that diversity, equity, and inclusion are reflected in funded research and the biomedical workforce.

Signalling mechanisms and cellular functions of SUMO.

Sumoylation is an essential post-translational modification that is catalysed by a small number of modifying enzymes but regulates thousands of target proteins in a dynamic manner. Small ubiquitin-like modifiers (SUMOs) can be attached to target proteins as one or more monomers or in the form of polymers of different types. Non-covalent readers recognize SUMO-modified proteins via SUMO interaction motifs. SUMO simultaneously modifies groups of functionally related proteins to regulate predominantly nuclear processes, including gene expression, the DNA damage response, RNA processing, cell cycle progression and proteostasis. Recent progress has increased our understanding of the cellular and pathophysiological roles of SUMO modifications, extending their functions to the regulation of immunity, pluripotency and nuclear body assembly in response to oxidative stress, which partly occurs through the recently characterized mechanism of liquid-liquid phase separation. Such progress in understanding the roles and regulation of sumoylation opens new avenues for the targeting of SUMO to treat disease, and indeed the first drug blocking sumoylation is currently under investigation in clinical trials as a possible anticancer agent.

A comprehensive compilation of SUMO proteomics.

Small ubiquitin-like modifiers (SUMOs) are essential for the regulation of several cellular processes and are potential therapeutic targets owing to their involvement in diseases such as cancer and Alzheimer disease. In the past decade, we have witnessed a rapid expansion of proteomic approaches for identifying sumoylated proteins, with recent advances in detecting site-specific sumoylation. In this Analysis, we combined all human SUMO proteomics data currently available into one cohesive database. We provide proteomic evidence for sumoylation of 3,617 proteins at 7,327 sumoylation sites, and insight into SUMO group modification by clustering the sumoylated proteins into functional networks. The data support sumoylation being a frequent protein modification (on par with other major protein modifications) with multiple nuclear functions, including in transcription, mRNA processing, DNA replication and the DNA-damage response.

Physical interactions between specifically regulated subpopulations of the MCM and RNR complexes prevent genetic instability.

The helicase MCM and the ribonucleotide reductase RNR are the complexes that provide the substrates (ssDNA templates and dNTPs, respectively) for DNA replication. Here, we demonstrate that MCM interacts physically with RNR and some of its regulators, including the kinase Dun1. These physical interactions encompass small subpopulations of MCM and RNR, are independent of the major subcellular locations of these two complexes, augment in response to DNA damage and, in the case of the Rnr2 and Rnr4 subunits of RNR, depend on Dun1. Partial disruption of the MCM/RNR interactions impairs the release of Rad52 -but not RPA-from the DNA repair centers despite the lesions are repaired, a phenotype that is associated with hypermutagenesis but not with alterations in the levels of dNTPs. These results suggest that a specifically regulated pool of MCM and RNR complexes plays non-canonical roles in genetic stability preventing persistent Rad52 centers and hypermutagenesis.

WWP2 ubiquitylates RNA polymerase II for DNA-PK-dependent transcription arrest and repair at DNA breaks.

DNA double-strand breaks (DSBs) at RNA polymerase II (RNAPII) transcribed genes lead to inhibition of transcription. The DNA-dependent protein kinase (DNA-PK) complex plays a pivotal role in transcription inhibition at DSBs by stimulating proteasome-dependent eviction of RNAPII at these lesions. How DNA-PK triggers RNAPII eviction to inhibit transcription at DSBs remains unclear. Here we show that the HECT E3 ubiquitin ligase WWP2 associates with components of the DNA-PK and RNAPII complexes and is recruited to DSBs at RNAPII transcribed genes. In response to DSBs, WWP2 targets the RNAPII subunit RPB1 for K48-linked ubiquitylation, thereby driving DNA-PK- and proteasome-dependent eviction of RNAPII. The lack of WWP2 or expression of nonubiquitylatable RPB1 abrogates the binding of nonhomologous end joining (NHEJ) factors, including DNA-PK and XRCC4/DNA ligase IV, and impairs DSB repair. These findings suggest that WWP2 operates in a DNA-PK-dependent shutoff circuitry for RNAPII clearance that promotes DSB repair by protecting the NHEJ machinery from collision with the transcription machinery.

A disease-associated XPA allele interferes with TFIIH binding and primarily affects transcription-coupled nucleotide excision repair.

XPA is a central scaffold protein that coordinates the assembly of repair complexes in the global genome (GG-NER) and transcription-coupled nucleotide excision repair (TC-NER) subpathways. Inactivating mutations in XPA cause xeroderma pigmentosum (XP), which is characterized by extreme UV sensitivity and a highly elevated skin cancer risk. Here, we describe two Dutch siblings in their late forties carrying a homozygous H244R substitution in the C-terminus of XPA. They present with mild cutaneous manifestations of XP without skin cancer but suffer from marked neurological features, including cerebellar ataxia. We show that the mutant XPA protein has a severely weakened interaction with the transcription factor IIH (TFIIH) complex leading to an impaired association of the mutant XPA and the downstream endonuclease ERCC1-XPF with NER complexes. Despite these defects, the patient-derived fibroblasts and reconstituted knockout cells carrying the XPA-H244R substitution show intermediate UV sensitivity and considerable levels of residual GG-NER (~50%), in line with the intrinsic properties and activities of the purified protein. By contrast, XPA-H244R cells are exquisitely sensitive to transcription-blocking DNA damage, show no detectable recovery of transcription after UV irradiation, and display a severe deficiency in TC-NER-associated unscheduled DNA synthesis. Our characterization of a new case of XPA deficiency that interferes with TFIIH binding and primarily affects the transcription-coupled subpathway of nucleotide excision repair, provides an explanation of the dominant neurological features in these patients, and reveals a specific role for the C-terminus of XPA in TC-NER.

A Chain of Events: Regulating Target Proteins by SUMO Polymers.

Small ubiquitin-like modifiers (SUMOs) regulate virtually all nuclear processes. The fate of the target protein is determined by the architecture of the attached SUMO protein, which can be of polymeric nature. Here, we highlight the multifunctional aspects of dynamic signal transduction by SUMO polymers. The SUMO-targeted ubiquitin ligases (STUbLs) RING-finger protein 4 (RNF4) and RNF111 recognize SUMO polymers in a chain-architecture-dependent manner, leading to the formation of hybrid chains, which could enable proteasomal destruction of proteins. Recent publications have highlighted essential roles for SUMO chain disassembly by the mammalian SUMO proteases SENP6 and SENP7 and the yeast SUMO protease Ulp2. SENP6 is particularly important for centromere assembly. These recent findings demonstrate the diversity of SUMO polymer signal transduction for proteolytic and nonproteolytic purposes.

Site-specific proteomic strategies to identify ubiquitin and SUMO modifications: Challenges and opportunities.

Ubiquitin and SUMO modify thousands of substrates to regulate most cellular processes. System-wide identification of ubiquitin and SUMO substrates provides global understanding of their cellular functions. In this review, we discuss the biological importance of site-specific modifications by ubiquitin and SUMO regulating the DNA damage response, protein quality control and cell cycle progression. Furthermore we discuss the machinery responsible for these modifications and methods to purify and identify ubiquitin and SUMO modified sites by mass spectrometry. We provide a framework to aid in the selection of appropriate purification, digestion and acquisition strategies suited to answer different biological questions. We highlight opportunities in the field for employing innovative technologies, as well as discuss challenges and long-standing questions in the field that are difficult to address with the currently available tools, emphasizing the need for further innovation.

PARP1 Links CHD2-Mediated Chromatin Expansion and H3.3 Deposition to DNA Repair by Non-homologous End-Joining.

The response to DNA double-strand breaks (DSBs) requires alterations in chromatin structure to promote the assembly of repair complexes on broken chromosomes. Non-homologous end-joining (NHEJ) is the dominant DSB repair pathway in human cells, but our understanding of how it operates in chromatin is limited. Here, we define a mechanism that plays a crucial role in regulating NHEJ in chromatin. This mechanism is initiated by DNA damage-associated poly(ADP-ribose) polymerase 1 (PARP1), which recruits the chromatin remodeler CHD2 through a poly(ADP-ribose)-binding domain. CHD2 in turn triggers rapid chromatin expansion and the deposition of histone variant H3.3 at sites of DNA damage. Importantly, we find that PARP1, CHD2, and H3.3 regulate the assembly of NHEJ complexes at broken chromosomes to promote efficient DNA repair. Together, these findings reveal a PARP1-dependent process that couples ATP-dependent chromatin remodeling with histone variant deposition at DSBs to facilitate NHEJ and safeguard genomic stability.

Exome sequencing links the SUMO protease SENP7 with fatal arthrogryposis multiplex congenita, early respiratory failure and neutropenia.

BACKGROUND: SUMOylation involves the attachment of small ubiquitin-like modifier (SUMO) proteins to specific lysine residues on thousands of substrates with target-specific effects on protein function. Sentrin-specific proteases (SENPs) are proteins involved in the maturation and deconjugation of SUMO. Specifically, SENP7 is responsible for processing polySUMO chains on targeted substrates including the heterochromatin protein 1α (HP1α). METHODS: We performed exome sequencing and segregation studies in a family with several infants presenting with an unidentified syndrome. RNA and protein expression studies were performed in fibroblasts available from one subject. RESULTS: We identified a kindred with four affected subjects presenting with a spectrum of findings including congenital arthrogryposis, no achievement of developmental milestones, early respiratory failure, neutropenia and recurrent infections. All died within four months after birth. Exome sequencing identified a homozygous stop gain variant in SENP7 c.1474C>T; p.(Gln492*) as the probable aetiology. The proband's fibroblasts demonstrated decreased mRNA expression. Protein expression studies showed significant protein dysregulation in total cell lysates and in the chromatin fraction. We found that HP1α levels as well as different histones and H3K9me3 were reduced in patient fibroblasts. These results support previous studies showing interaction between SENP7 and HP1α, and suggest loss of SENP7 leads to reduced heterochromatin condensation and subsequent aberrant gene expression. CONCLUSION: Our results suggest a critical role for SENP7 in nervous system development, haematopoiesis and immune function in humans.

THO complex deficiency impairs DNA double-strand break repair via the RNA surveillance kinase SMG-1.

The integrity and proper expression of genomes are safeguarded by DNA and RNA surveillance pathways. While many RNA surveillance factors have additional functions in the nucleus, little is known about the incidence and physiological impact of converging RNA and DNA signals. Here, using genetic screens and genome-wide analyses, we identified unforeseen SMG-1-dependent crosstalk between RNA surveillance and DNA repair in living animals. Defects in RNA processing, due to viable THO complex or PNN-1 mutations, induce a shift in DNA repair in dividing and non-dividing tissues. Loss of SMG-1, an ATM/ATR-like kinase central to RNA surveillance by nonsense-mediated decay (NMD), restores DNA repair and radio-resistance in THO-deficient animals. Mechanistically, we find SMG-1 and its downstream target SMG-2/UPF1, but not NMD per se, to suppress DNA repair by non-homologous end-joining in favour of single strand annealing. We postulate that moonlighting proteins create short-circuits in vivo, allowing aberrant RNA to redirect DNA repair.

Tissue oxygenation stabilizes neovessels and mitigates hemorrhages in human atherosclerosis-induced angiogenesis.

Progression of atherosclerosis is associated with a maladaptive form of angiogenesis which contributes to intraplaque hemorrhage and plaque disruption. Hypoxia has been implicated in mechanisms of angiogenic neovessel fragility and atherosclerotic plaque destabilization. We used ex vivo and in vivo models to characterize the effect of oxygen (O2) on the formation, stability and tendency to bleed of human plaque-induced neovessels. Plaque explants potently stimulated the ex vivo angiogenic response of rat aortic rings at atmospheric O2 levels. Severe hypoxia (1% O2) inhibited plaque-induced angiogenesis and pericyte recruitment causing neovessel breakdown, whereas increasing O2 levels dose dependently enhanced pericyte numbers and neovessel stability. Plaque fragments implanted subcutaneously with or without aortic rings in SCID mice stimulated the host angiogenic response with plaques causing minimal or no hemorrhages and plaques co-implanted with aortic rings causing marked hemorrhages. Plaque/aortic ring-induced hemorrhages were reduced in mice exposed to moderate hyperoxia (50% O2). Hyperoxia downregulated expression of the hypoxia-sensitive genes Ca9, Ca12 and VegfA and increased influx into implants of mesenchymal cells reactive for the pericyte marker NG2. In both ex vivo and in vivo models, O2 promoted expression of vasostabilizing genes required for pericyte recruitment (Angpt1, Pdgfb), basement membrane assembly (Col4A1), and tight junction formation (Cldn5 and/or Ocln). Our results suggest that formation of neovessels that are stable, pericyte-coated, and resistant to bleeding requires adequate tissue oxygenation. Understanding the mechanisms by which O2 stabilizes neovessels and mitigates neovessel bleeding may lead to new therapies for the prevention of atherosclerosis complications.

In-Plate Chemical Synthesis of Isopeptide-Linked SUMOylated Peptide Fluorescence Polarization Reagents for High-Throughput Screening of SENP Preferences.

Small ubiquitin-like modifiers (SUMOs) are conjugated to protein substrates in cells to regulate their function. The attachment of SUMO family members SUMO1-3 to substrate proteins is reversed by specific isopeptidases called SENPs (sentrin-specific protease). Whereas SENPs are SUMO-isoform or linkage type specific, comprehensive analysis is missing. Furthermore, the underlying mechanism of SENP linkage specificity remains unclear. We present a high-throughput synthesis of 83 isopeptide-linked SUMO-based fluorescence polarization reagents to study enzyme preferences. The assay reagents were synthesized via a native chemical ligation-desulfurization protocol between 11-mer peptides containing a γ-thiolysine and a SUMO3 thioester. Subsequently, five recombinantly expressed SENPs were screened using these assay reagents to reveal their deconjugation activity and substrate preferences. In general, we observed that SENP1 is the most active and nonselective SENP while SENP6 and SENP7 show the least activity. Furthermore, SENPs differentially process peptides derived from SUMO1-3, who form a minimalistic representation of diSUMO chains. To validate our findings, five distinct isopeptide-linked diSUMO chains were chemically synthesized and proteolysis was monitored using a gel-based read-out.

Theoretical Feasibility of Using Arch Branched Endograft Devices for Repair of Post Type A Aortic Dissection in Patients With Prior Ascending Aortic Replacement.

PURPOSE: Ascending aortic replacement is a common emergency procedure for treating acute type A aortic dissection. Secondary open or endovascular interventions for residual arch pathologies is difficult because of adhesions, short prosthetic grafts, and distorted anatomies. Aortic arch branched stent grafts have emerged as a potential solution for these patients if they have suitable anatomical conditions. This study aimed to evaluate the theoretical anatomical and technical feasibility of 2 currently used aortic arch branch endografts in patients who had prior replacement of the ascending aorta. MATERIALS AND METHODS: All patients who had a prosthetic ascending aortic or hemiarch replacement for acute type A dissection in a single institution between January 2013 and December 2018 were included. Contrast computed tomography images on the most recent follow-up were analyzed on a 3-dimensional workstation. Morphological parameters were measured individually for the ascending aorta, aortic arch, supra-aortic branches, and access iliac arteries. The computed tomography scan of each patient was individually evaluated for anatomical suitability for the arch branched and double-branch devices according to set selection criteria. RESULTS: Computed tomography images of 56 patients (median age of 57 years, 45 males) were reviewed. Based on our evaluation, 26 patients (46.4%) were good candidates for an endovascular arch branched device. It would be feasible for 13 patients (23.2%), but prudent preoperative planning was required due to complicated anatomy. The other 17 patients (30.4%) were unsuitable because they met at least 1 exclusion criterion. Short prosthetic grafts, extreme graft angulations, and extensive dissections in the supra-aortic branches were the main reasons for exclusion. CONCLUSION: Endovascular repair using arch branched endografts is feasible in patients with prior ascending aortic arch or hemiarch replacement for acute type A aortic dissection. The most common anatomical conditions that may influence the feasibility of the arch branched endograft procedure include insufficient proximal seal length, severe angulation of the graft, and extensive aortic dissection within the supra-aortic vessels.

Sensory weighting of position and force feedback during pinching.

Human hands are complex biomechanical systems that allow for dexterous tasks with many degrees of freedom. Coordination of the fingers is essential for many activities of daily living and involves integrating sensory signals. During this sensory integration, the central nervous system deals with the uncertainty of sensory signals. When handling compliant objects, force and position are related. Interactions with stiff objects result in reduced position changes and increased force changes compared to compliant objects. Literature has shown sensory integration of force and position at the shoulder. Nevertheless, differences in sensory requirements between proximal and distal joints may lead to different proprioceptive representations, hence findings at proximal joints cannot be directly transferred to distal joints, such as the digits. Here, we investigate the sensory integration of force and position during pinching. A haptic manipulator rendered a virtual spring with adjustable stiffness between the index finger and the thumb. Participants had to blindly reproduce a force against the spring. In both visual reference trials and blind reproduction trials, the relation between pinch force and spring compression was constant. However, by covertly changing the spring characteristics in catch trials into an adjusted force-position relation, the participants' weighting of force and position could be revealed. In agreement with previous studies on the shoulder, participants relied more on force sense in trials with higher stiffness. This study demonstrated stiffness-dependent sensory integration of force and position feedback during pinching.

Wang-Landau sampling of lattice multiblock copolymers.

Synthetic multiblock copolymers are an interesting class of polymeric chains and have emerged as promising materials to mimic the function of complex biomolecules. In this work, we use Wang-Landau sampling to study sequences of multiblock (AnBn)m copolymers on the simple cubic lattice, where n represents the block length and m represents the number of blocks. We first compare to the thermodynamic and structural properties of four sequences previously studied in the continuum [W. Wang et al., J. Chem. Phys. 141, 244907 (2014)] to observe the differences that arise during the collapse process. We then focus on the structural transitions that occur at temperatures below the coil-to-globule transition in the lattice. Moreover, by studying additional sequences, we detail the relationship between the block length, number of blocks, and, thus, overall polymer length with respect to said structural transitions. Finally, we observe how the formation and shape of a ground state core of the more strongly interacting monomer type affect the procession of structural changes that occurs as temperature increases.

Ultrasound vector flow imaging during veno-arterial extracorporeal membrane oxygenation in a thoracic aorta model.

In veno-arterial extracorporeal membrane oxygenation (VA-ECMO) treatment, the mixing zone is a key hemodynamic factor that determines the efficacy of the treatment. This study aimed to evaluate the applicability of a novel ultrasound technique called vector flow imaging (VFI) for visualizing complex flow patterns in an aorta phantom under VA-ECMO settings. VFI experiments were performed to image aortic hemodynamics under VA-ECMO treatment simulated in an anthropomorphic thoracic aorta phantom using a pulsatile pump (cardiac output: 2.7 L/min) and an ECMO pump with two different flow rates, 0.35 L/min and 1.0 L/min. The cardiac cycle of hemodynamics in the ascending aorta, aortic arch, and descending aorta was visualized, and the spatio-temporal dynamics of flow vectors were analyzed. VFI successfully visualized dynamic flow patterns in the aorta phantom. When the flow rate of the ECMO pump increased, ECMO flow was more dominant than cardiac output in the diastole phase, and the speed of cardiac output was suppressed in the systole phase. Vortex flow patterns were also detected in the ascending aorta and the arch under both ECMO flow rate conditions. The VFI technique may provide new insights into aortic hemodynamics and facilitates effective and safe VA-ECMO treatment.

Examining the role of intrinsic and reflexive contributions to ankle joint hyper-resistance treated with botulinum toxin-A.

BACKGROUND: Spasticity, i.e. stretch hyperreflexia, increases joint resistance similar to symptoms like hypertonia and contractures. Botulinum neurotoxin-A (BoNT-A) injections are a widely used intervention to reduce spasticity. BoNT-A effects on spasticity are poorly understood, because clinical measures, e.g. modified Ashworth scale (MAS), cannot differentiate between the symptoms affecting joint resistance. This paper distinguishes the contributions of the reflexive and intrinsic pathways to ankle joint hyper-resistance for participants treated with BoNT-A injections. We hypothesized that the overall joint resistance and reflexive contribution decrease 6 weeks after injection, while returning close to baseline after 12 weeks. METHODS: Nine participants with spasticity after spinal cord injury or after stroke were evaluated across three sessions: 0, 6 and 12 weeks after BoNT-A injection in the calf muscles. Evaluation included clinical measures (MAS, Tardieu Scale) and motorized instrumented assessment using the instrumented spasticity test (SPAT) and parallel-cascade (PC) system identification. Assessments included measures for: (1) overall resistance from MAS and fast velocity SPAT; (2) reflexive resistance contribution from Tardieu Scale, difference between fast and slow velocity SPAT and PC reflexive gain; and (3) intrinsic resistance contribution from slow velocity SPAT and PC intrinsic stiffness/damping. RESULTS: Individually, the hypothesized BoNT-A effect, the combination of a reduced resistance (week 6) and return towards baseline (week 12), was observed in the MAS (5 participants), fast velocity SPAT (2 participants), Tardieu Scale (2 participants), SPAT (1 participant) and reflexive gain (4 participants). On group-level, the hypothesis was only confirmed for the MAS, which showed a significant resistance reduction at week 6. All instrumented measures were strongly correlated when quantifying the same resistance contribution. CONCLUSION: At group-level, the expected joint resistance reduction due to BoNT-A injections was only observed in the MAS (overall resistance). This observed reduction could not be attributed to an unambiguous group-level reduction of the reflexive resistance contribution, as no instrumented measure confirmed the hypothesis. Validity of the instrumented measures was supported through a strong association between different assessment methods. Therefore, further quantification of the individual contributions to joint resistance changes using instrumented measures across a large sample size are essential to understand the heterogeneous response to BoNT-A injections.

Targeting pancreatic cancer by TAK-981: a SUMOylation inhibitor that activates the immune system and blocks cancer cell cycle progression in a preclinical model.

OBJECTIVE: Pancreatic ductal adenocarcinoma (PDAC) has the characteristics of high-density desmoplastic stroma, a distinctive immunosuppressive microenvironment and is profoundly resistant to all forms of chemotherapy and immunotherapy, leading to a 5-year survival rate of 9%. Our study aims to add novel small molecule therapeutics for the treatment of PDAC. DESIGN: We have studied whether TAK-981, a novel highly selective and potent small molecule inhibitor of the small ubiquitin like modifier (SUMO) activating enzyme E1 could be used to treat a preclinical syngeneic PDAC mouse model and we have studied the mode of action of TAK-981. RESULTS: We found that SUMOylation, a reversible post-translational modification required for cell cycle progression, is increased in PDAC patient samples compared with normal pancreatic tissue. TAK-981 decreased SUMOylation in PDAC cells at the nanomolar range, thereby causing a G2/M cell cycle arrest, mitotic failure and chromosomal segregation defects. TAK-981 efficiently limited tumour burden in the KPC3 syngeneic mouse model without evidence of systemic toxicity. In vivo treatment with TAK-981 enhanced the proportions of activated CD8 T cells and natural killer (NK) cells but transiently decreased B cell numbers in tumour, peripheral blood, spleen and lymph nodes. Single cell RNA sequencing revealed activation of the interferon response on TAK-981 treatment in lymphocytes including T, B and NK cells. TAK-981 treatment of CD8 T cells ex vivo induced activation of STAT1 and interferon target genes. CONCLUSION: Our findings indicate that pharmacological inhibition of the SUMO pathway represents a potential strategy to target PDAC via a dual mechanism: inhibiting cancer cell cycle progression and activating anti-tumour immunity by inducing interferon signalling.

Guiding Mitotic Progression by Crosstalk between Post-translational Modifications.

Cell division is tightly regulated to disentangle copied chromosomes in an orderly manner and prevent loss of genome integrity. During mitosis, transcriptional activity is limited and post-translational modifications (PTMs) are responsible for functional protein regulation. Essential mitotic regulators, including polo-like kinase 1 (PLK1) and cyclin-dependent kinases (CDK), as well as the anaphase-promoting complex/cyclosome (APC/C), are members of the enzymatic machinery responsible for protein modification. Interestingly, communication between PTMs ensures the essential tight and timely control during all consecutive phases of mitosis. Here, we present an overview of current concepts and understanding of crosstalk between PTMs regulating mitotic progression.